Identification of Some Active Proteins in the Process of Hen Eggshell Formation

Abstract

The hen eggshell is formed in a three-stage sequential process, namely, the initial stage (nucleation of calcite crystals), the active growth phase (linear deposition), and the terminal stage (inhibition of crystal formation). During these phases, different proteins are sequentially expressed in the uterine fluid. Some of them are thought to regulate the shell mineralization and particularly crystal growth. To identify proteins that are actively involved in this process, we have analyzed and compared the effects on CaCO3 precipitation in vitro of some commercial egg white proteins, some noncommercial purified fractions of the eggshell organic matrix and, also of uterine fluids extracted from the hen uterus during each one of the stages of calcification. Uterine fluids strongly increased the nucleation density with respect to commercial proteins and purified fractions of the shell. A few identified acidic proteins (ovocleidin-17, ovocalyxin-32, osteopontin and ovocleidin-116) showed a strong affinity for the CaCO3 surfaces and were selectively removed from the solution during its precipitation. These proteins may have an active role on CaCO3 growth, aggregation, and inhibition. Other proteins, with very different isoelectric points, seem to regulate the chemical environment in which the precipitation takes place, that is, by buffering the pH favoring crystal growth as the couple ovocleidin-17 and ovocalyxin-21.

Structural and physical changes in the hen's eggshell in response to the inclusion of dietary organic minerals

“…The spherical and rhombohedral forms of calcite identified in the current study conform to the crystal growth patterns described by Hernandez-Hernandez et al (2008b). In their investigations the authors demonstrated the role of specific protein complexes in the exclusive selection of calcite as the dominant polymorph in the avian eggshell.…”

Protein constituents of the eggshell: eggshell-specific matrix proteins

“…In contrast, the total uterine fluid harvested at the terminal stage of calcification inhibits calcite precipitation [19]. In agreement with these observations, partially purified eggshell matrix proteins inhibit calcium carbonate precipitation and alter patterns of calcite crystal growth, leading to morphological modifications of rhombohedric calcite crystals grown in vitro [19,42]. Studies to investigate the effect of purified eggshell matrix proteins on calcium carbonate crystallization and crystal growth are described in the next section.…”

“…A number of experimental observations support the role of the eggshell matrix proteins in determining the fabric of the eggshell and therefore influencing its resulting mechanical properties. Proteins in the uterine fluid modify the kinetics of calcium carbonate precipitation in vitro [41,42]. The lag time for calcium carbonate precipitation is reduced by the uterine fluid from the initial and growth stages of eggshell mineralization, suggesting that these matrix precursors promote crystal nucleation.…”

“…This hypothesis originated from the observations of morphological changes in calcite crystals by uterine fluid collected during the terminal phase of calcification and the location of OCX-32 in the mineral pellet after its precipitation with calcium carbonate in vitro from fresh uterine fluid [41,42]. Studies with purified OCX-32 are necessary to investigate this possibility.…”

Ultrastructure of avian eggshell during resorption following egg fertilization

“…Mineral textural analyses of avian eggshell using Xray analysis and polarized light show no evidence that ACC is present in completed, laid calcitic eggshell (Rodriguez-Navarro et al, 2007). More recently, this same group found no ACC phase in crystal growth studies in vitro (Hemandez-Hemandez et al, 2008), although others growing calcium carbonate in vitro observed its transient presence when quail eggshell proteins were present (Lakshminarayanan et al, 2006). Despite this for avian eggshell, in the case of calcium carbonate sea urchin spine and larval skeleton, for example, higher resolution studies show that the ''single crystal" biominerals are actually a mosaic of domains delimited by distinct or occluded organic proteinaceous layers (Berman et al, 1988(Berman et al, , 1990 and contain transient ACC (Wilt, 1999(Wilt, , 2002.…”